106 USING A BUOYANT DENSITY GRADIENT AND NILE RED STAINING TO EVALUATE THE LIPID CONTENT OF BOS TAURUS AND BOS INDICUS OOCYTES
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Résumé
Bos indicus embryos have a lower survival rate compared with Bos taurus after cryopreservation. It has been hypothesized that the lower survival rate is due to higher intracellular lipid content. The objective of this study was to determine if there is a difference in intracellular lipid content of oocytes from mature purebred Brahman and Angus cows. Donor females used in the study were maintained on pasture prior to the onset of the experiment and on a grain-supplemented hay ration during the study. Oocytes were collected from cows at 30-day intervals (3 aspirations/donor) by transvaginal ultrasound-guided oocyte aspirations (TUGA). Mature oocytes were evaluated using a sucrose step gradient procedure and Nile Red staining. FSH (Folltropin-V®; Bioniche Animal Health, Beltsville, Ontario, Canada) administration began on Day 4 of the estrous cycle (estrus = Day 0) twice daily for 3 days in decreasing doses (Brahman 232 mg and Angus 280 mg total), and on Day 8 oocytes were recovered. The mean number of follicles aspirated/donor and oocytes recovered/donor were 20 and 16.61 oocytes for the Brahman donors (n = 6) and 12 follicles and 7.06 oocytes/donor for the Angus donors (n = 10). Oocytes (individual donor basis) were then incubated in TCM-199 supplemented with 10% fetal bovine serum + bLH and bFSH (0.01 U mL-1) at 38.5°C. After 20 h, mature oocytes were denuded by vortexing for 3 min in HEPS + BSA (4 mg mL-1). Buoyancy was tested for individual mature oocytes using a sucrose step density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% sucrose (indicating high lipids) to 35% sucrose (indicating lower lipids). Oocytes recovered from the sucrose were fixed for 24 h in paraformaldehyde for evaluation with Nile Red stain. Oocytes were stained for 24 h, and then placed in Prolong® Gold (Invitrogen, Carlsbad, CA, USA) and evaluated under fluorescence. Oocytes images were evaluated using a Scion Image camera (Scion Corp., Frederick, MD, USA) to calculate mean (± SE) Nile Red units (NRU) (higher NRU = higher lipid content). Treatment groups were analyzed using one-way ANOVA. In summary, Brahman M-II oocytes had significantly lower (P = 0.05) buoyant density, with a significantly higher mean NRU score, when compared with oocytes harvested from Angus donors (Table 1). Based on these results, Brahman oocytes have a higher intracellular lipid content then Angus oocytes. Table 1. Percent sucrose levels and Nile Red units for bovine oocytes from three replicates per donor
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Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,002 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découle